During intracellular transport, proteins destined for the plasma membrane, secretory vesicles and lysosomes must be sorted from one another within the Golgi complex, and sent to their appropriate addresses. The long term goal of this research is to elucidate the molecular mechanisms by which proteins are targeted to specific and distinct intracellular destinations. We study the targeting of proteins to lysosomes. Soluble lysosomal enzymes acquire a mannose 6-phosphate tag within the Golgi complex; intracellular receptors (MPRs) recognize these proteins, and facilitate their transport to lysosomes. We have reconstituted the recycling of MPRs from late endosomes to the trans Golgi network (TGN) in crude extracts. Our most significant accomplishment of the previous funding period was the discovery that rab9, a small ras-like GTPase, facilitates this transport reaction, both in vitro and in vivo. The present application describes experiments designed to continue our molecular dissection of MPR trafficking. The specific aims of this proposal are: 1. Elucidate the mechanism by which rab9 protein is localized specifically to late endosomes using an in vitro system which reconstitutes organelle-specific localization of rab 9 protein in vitro. 2. Dissect our in vitro assay into vesicle budding and targeting subreactions and establish a functional connection between rab recruitment and subsequent MPR transport from late endosomes to the TGN. 3. Test the validity of the SNARE Hypothesis for endosome-TGN transport by establishing whether known SNARE constituents participate in MPR recycling from late endosomes to the TGN in vitro. Use a functional assay for rab9 protein to attempt to link the SNARE machinery with rab protein function. 4. Investigate the role of ARF in endosome-to-TGN transport to aid in the identification of the putative coat proteins and transport vesicles (or tubules) responsible for this transport step. It is hoped that these experiments will provide new molecular detail as to the mechanism by which proteins are delivered from one membrane-bound compartment to another.